Increased insulin-like growth factor binding protein-2 (IGFBP-2) gene expression and protein production lead to high IGFBP-2 content in malignant ovarian cyst fluid

H Kanety,J Menczer,I Goldberg,J Kopolovic,A Karasik,J Ravia,M Kattan

doi:10.1038/bjc.1996.206

Abstract

Expression of insulin-like growth factor-I (IGF-I), its receptor and IGF-binding proteins (IGFBPs) by ovarian cancer cells and its mitogenic effect on these cells in vitro, suggest that IGF-I may have a role in regulation of human ovarian cancer. We have recently shown IGFBP-2 to be markedly elevated in malignant ovarian cyst fluid in vivo. To identify the origin of increased IGFBP-2 in these cyst fluids, the gene expression and protein content of IGFBP-2 were investigated in 14 malignant and four benign epithelial ovarian neoplasms. IGFBP-2 mRNA was detected in all ovarian specimens and was 2- to 30-fold higher in malignant than in benign neoplasms. Within the malignant tissues IGFBP-2 mRNA levels correlated with the aggressiveness of the tumour and were higher in invasive tumours than in those with borderline pathology. Southern blot analysis revealed no amplification of IGFBP-2 gene in the DNA samples from ovarian tumours regardless of their nature. IGFBP-2 was the major binding protein in tissue extracts, as measured by both Western ligand blotting and immunoblotting, and was significantly higher in malignant than in benign neoplasms. These findings were further supported by immunohistochemical detection of IGFBP-2 in tumour sections. Our data suggest that increased local production by the tumour in vivo is responsible for the increased IGFBP-2 levels in the cyst fluid bathing the ovarian malignancy. This may represent an autocrine regulatory mechanism for IGF-I proliferative effect of ovarian cancer.

Highlights

We have recently reported Insulin-like growth factors' (IGFs)-binding proteins (IGFBPs)-2 to be significantly higher in cyst fluid from invasive malignant than from benign epithelial ovarian neoplasms (Karasik et al, 1994)
IGFBPs in protein extracts prepared from primary benign and malignant epithelial ovarian neoplasms were evaluated by Western ligand blotting and immunoblotting
To evaluate further the origin of the high IGFBP-2 levels observed in ovarian malignant neoplasms, RNA isolated from benign (Ben, n = 4), borderline malignant (B, n = 3), invasive primary (I, n = 5) and metastatic (M, n = 6) ovarian neoplasms was assayed for IGFBP-2 gene expression

Summary

Materials and methods

Tumours were dissected, frozen immediately in liquid nitrogen and kept at -80°C until analysed. Paraffin-embedded tissue sections (4 Mrm-thick) were deparaffinised, hydrated through xylene-graded alcohol series, treated with 3% hydrogen peroxide (H202) to neutralise endogenous peroxidase, washed in Tris buffer (pH 7.6) and incubated with rabbit antiserum against bovine IGFBP-2 (1:200) or rabbit antiserum against human IGFBP-3 (1:200) for 30 min at room temperature. Sections were washed extensively and incubated with biotinylated anti-rabbit IgG for 30 min, followed by incubation for an additional 30 min with streptavidin-conjugated horseradish peroxidase using a commercial kit (Dako, Cappinteria, CA, USA) according to the manufacturer's directions. The cytoplasm of both the benign and malignant epithelium stained positive for IGFBP-2. The stroma surrounding these structures did not stain positive for either IGFBP-2 (Figure 2) or IGFBP-3 (not shown)

Results

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